Protocols for Immunohistochemistry
Our staining kits are provided with complete protocols for use. This page lists the steps involved in using selected Histostain™ kits and the time required for each step. It also contains protocols for removing endogenous peroxidase, digesting tissue and antigen recovery.
Removal of Endogenous Peroxidase Activity
- Procedure 1: (Use for paraffin-embedded sections. Not for frozen sections.) Prepare peroxidase blocking solution by adding one part 30% hydrogen peroxide to nine parts methanol. Submerge slides in this solution for 10 minutes. Wash three times with PBS.
- Procedure 2: (May be used with frozen or paraffin-embedded sections.)
Treat slides with Peroxo-block™ for 45 seconds. Wash immediately.
Removal of residual biotin, avidin or streptavidin activity
- Residual Biotin: Incubate section with the avidin solution from our Avidin/Biotin Blocking Kit. Wash. Incubate with the d-biotin solution from the kit. Wash thoroughly.
- Residual Avidin or Streptavidin: Incubate section with the d-Biotin solution from our Avidin/Biotin Blocking Kit. Wash thoroughly.
Heat Induced Epitope Retrieval
The epitope retrieval procedure is used to reverse the loss of antigenicity that occurs with some epitopes in formalin-fixed, paraffin-embedded tissues. We have found that some antibodies require Antigen Recovery (e.g., estrogen receptor, and Ki-67 antibodies), while the staining of many other antibodies will be enhanced by Antigen Recovery (e.g., S-100, cytokeratin, and synaptophysin antibodies). No licensing fee is required for using Antigen Recovery.
A. Materials
- Hot plate
- 1 liter glass beaker
- 0.01 M citrate buffer, pH 6.0 (No. 00-5000)
- EDTA Solution (Cat. No. 00-5500 for antibodies that require EDTA instead of citrate buffer)
- PBS
B. Protocol
- Mount tissues should on silane or poly-L-Lysine coated slides, or slides coated with HistoGrip™.
- Deparaffinize slides and, if desired, perform avidin/biotin blocking and endogenous enzyme quenching (see above).
- Wash slides with distilled water 3 times for 2 minutes each.
- Put the slides in a slide rack and place it in a 1 liter glass beaker (Pyrex) containing 500 ml of 0.01 M citrate buffer.
- Place beaker on hot plate. Heat the solution until it boils and keep it boiling for 10 minutes.
- After heating, remove beaker from the hot plate and allow it to cool down for at least 10-20 minutes at room temperature.
- Rinse slides with PBS and start the immunostaining protocol.
Tissue Pre-digestion
Tissue digestion recommendations for all predilute and 50x concentrated IHC antibodies are given in our Anatomical Pathology Catalog. Tissues should be mounted on slides using HistoGrip™, Para-Pen , Fro-Pen, silane, or poly-l-lysine.
- Deparaffinize, rehydrate, and quench endogenous enzyme activity (if necessary).
- Wash slides with distilled water 3 times for 2 minutes each.
- Incubate sections with proteolytic enzyme at 37oC. Incubation time should be determined for each application (for digestion reagents standardized for 10 min incubations, inquire about Digest-All™
- Rinse slides with PBS 3 times for 2 minutes each and continue the immunostaining procedure.